Immunization has been used for over a hundred years to protect humans and animals against disease. The premise of traditional immunization is that the most effective immune responses to an antigen, or a pathogen containing the antigen, occur after an individual is challenged with that same antigen two or more times. This phenomena is called immunological memory or a secondary immune response. When the immunization is successful, the individual is protected from the effects of the pathogen from which the antigen was derived.
For example, once an individual is successfully immunized with an antigen derived from a bacterial organism, the immune system in that individual is primed and ready to respond to that bacteria when it is encountered. Successful immunization requires that the antigen is located on an area of the bacteria that is accessible to the individual's immune system. When successful, the immune system responds, the bacteria is killed, contained, neutralized, or otherwise cleared from the body, and little or no disease results from the infection by the bacterial organism. The key to this protection is that immunization with the antigen must occur prior to the exposure to the bacterial organism from which the antigen is derived.
Accordingly, the traditional immunization process generally includes injecting an antigen into an individual, waiting an appropriate amount of time, and allowing the individual to mount an immune response. The time required for mounting an immune response is between approximately two weeks and several months for most antigens. In most cases, a booster administration of the antigen is required to maintain the immune response. This booster is normally given weeks or months after the initial administration of the antigen.
Therefore, traditional immunization is highly successful at providing protection if given several months in advance of exposure to an antigen, or pathogen, but traditional immunization is of little use when an individual is exposed to a new antigen to which the individual has not been previously exposed and an immediate effective immune response is required. A good example of such a situation is military troops in need of protection from bioterrorism agents. While a population of individuals can be vaccinated against agents of bioterrorism in advance of any potential exposure to the agents, traditional vaccination is not a simple answer. Traditional vaccination of a population creates harmful reactions in some persons and there is potential that the population may never be exposed to the agent, yet risks were taken. Additionally, a government cannot logistically develop, produce and vaccinate essential personnel with vaccines for every possible agent of bioterrorism. Compositions are needed that can be administered either immediately before, or even after, an individual's contact, or suspected contact, with a pathogen, which administration allows for the generation of an immediate protective or effective immune response in the individual.
Immunity linkers and universal immunogens have been previously constructed that provide a substantially immediate immunity such as those described in U.S. Patent Publication 20030017165 and 20040146515 incorporated herein by reference in their entirety. These previously described immunity linkers may incorporate aptamer nucleic acids as target binding elements. One disadvantage of using aptamer nucleic acids as a target binding site, is that nucleic acid molecules are subject to nuclease degradation. This reduces the half-life of these molecules, and by extension, the duration of the therapeutic benefit they provide.
Previous studies have shown that modified polynucleotides may be somewhat resistant to nuclease degradation. Modification of oligonucleotides such as by thiolation of the phosphoryl oxygens of the oligonucleotids can confer nuclease resistance (Gorenstein (Farschtschi, N. and Gorenstein, D. G., Tetrahedron Lett. (1988) 29:6843, and Nielsen, et al., Tetrahedron Lett. (1988) 29:291). Various backbone modifications such as the phosphorothioates and phosphorodithioates render the agents more nuclease-resistant. (Verma and Eckstein; Annu Rev Biochem, 1998 67:99-134). Unfortunately, oligonucleotides possessing high thiophosphate backbone substitutions appear to be “stickier” toward proteins than normal phosphate esters, attributable to non-specific interactions possibly based on the charge characteristics of the sulfonated nucleotides. The increased stickiness of thiolated ODNs results in loss of specificity, thus, defeating the promise of specific targeting offered by aptamer technology. Loss of specificity is critical in DNA binding proteins-DNA interactions, because most of the direct contacts between the proteins and their DNA binding sites are to the phosphate groups. As a further complication, it has been found that certain thiosubstitution can lead to structural perturbations in the structure of the duplex (Cho, et al., J. Biomol. Struct. Dyn. (1993) 11, 685-702). Therefore limited thiolation of nucleotides is possible to increase nuclease resistance, however this still does not provide appreciable circulating half-life for therapeutic applications such as in vivo administration, without sacrificing binding specificity.
What is therefore needed are immunity linkers having aptamer binding sites that are stabilized by including substantially all phosphorothioates or phosphorodithioates in the polynucleotide backbone, without reduced binding specificity.